PROJECT SUMMARY The goal of this proposal is to describe a 4 year plan for the PI, Christina Hickey, MD, to prepare for her independence as a physician scientist studying key epithelial cell signaling pathways triggered by commensal microbes that affect the development of inflammatory bowel disease. The PI graduated with distinction from Stanford University with a degree in Chemistry. She then obtained her medical degree from Washington University School of Medicine and completed her residency and pediatric gastroenterology fellowship at St. Louis Children's Hospital. During her fellowship, she published a first author paper with her current mentor, Dr. Thaddeus Stappenbeck, with the key finding that Bacteroides thetaiotaomicron (B. theta) outer membrane vesicles (OMVs) access immune cells in the intestine in an inflammatory bowel disease (IBD) mouse model. These findings serve as the basis for the aims in this proposal. Washington University School of Medicine and the Department of Pediatrics are exemplary locations for Dr. Hickey to start her research career with their collective longstanding history of NIH funded researchers and breadth and depth of resources from state of the art imaging centers to histological cores. In addition, there is frequent crosstalk and collaboration between departments, as Dr. Hickey's impressive mentoring committee and set of collaborators suggest, combining the expertise from the departments of microbiology (Drs. Martens, Beatty, and Tarr), immunology (Drs. Virgin and Allen), pathology (Dr. Stappenbeck), cell biology (Drs. Hou and Stahl) and gastroenterology (Dr. Tarr). The PI's mentor Dr. Stappenbeck is a leader in the field of intestinal host-microbial interactions; he is the ideal mentor for Dr. Hickey because of his keen ability to probe significant questions about pathogenesis, his solid track record in training young scientists, and his skill in writing, presentations, and troubleshooting projects. Furthermore, the Stappenbeck laboratory in which she is training is a hub of creative intellectual energy, composed of multiple post-doctoral fellows, graduate students, and technicians working on a broad range of projects. Dr. Hickey will also have multiple opportunities to present her work at Washington University and in the broader scientific community. The PI plans to take graduate level classes to enhance her knowledge in cellular biology and genomics. Finally, she will develop key skills in the labs of her collaborators and mentoring committee members. At the end of the 4 year award period, the PI will have accumulated the knowledge and skills she requires to become a successful physician scientist with an expertise in epithelial- commensal interactions. The PI's immediate goal is to fulfill the aims in this proposal. While it is clear that commensal microbial interactions with the intestinal epithelium are important for the development of pathogenesis, these interactions are poorly defined. In particular, she has recently shown that B. theta, a commensal organism, releases outer membrane vesicles that access the host immune cells on the basolateral side of the epithelium, indicating they have mechanisms to cross the epithelial barrier. Previous studies examining this host-microbial interface have utilized cancer cell lines and pathogens, which are not ideal tools to delineate this process. In this application, the PI proposes to utilize primary cell lines derived from genetic mouse models and OMVs isolated from the commensal microbe B. theta to probe the following: 1) the mechanism of epithelial internalization of B. theta OMVs, 2) the cell signaling pathways triggered by this internalization, and 3) the process by which OMVs pass through the epithelial layer. Based on her preliminary data, she hypothesizes that epithelial cells internalize B. theta OMVs via caveolin mediated lipid rafts, and the OMVs trigger epithelial signaling pathways to upregulate the internalization of additional OMVs, presumably to process them as a defense mechanism (Aim I). However, her preliminary findings also suggest that OMVs still traverse the epithelial layer, likely through defective tight junctions (Aim II). With the mechanisms uncovered in these aims, she will be ideally poised to fulfill her long- term goal of starting her own independent laboratory. Her lab's initial focus will be to determine the complete pathways of these mechanisms. Ultimately, she aims to translate her findings to human samples, which is critical for the development of more targeted therapies for inflammatory bowel disease.